PROJECT SUMMARY/ABSTRACT This renewal proposal explores molecular mechanisms that regulate alternative pre-mRNA splicing in differentiating erythroid cells, employing as a model the stage-specific activation of protein 4.1R exon 16 (E16) splicing that occurs in late erythoblasts and is essential for red cell membrane structural organization and function. Regulation of alternative splicing is a complex process involving combinatorial interactions among multiple components acting on the exon itself and on its flanking introns. Using a three exon 4.1R minigene as a splicing reporter for E16 splicing efficiency, this proposal will focus on analysis of intronic cis-regulatory sequences and splicing factor proteins that mediate splicing decisions. Special emphasis will be placed on the role of Fox-2, a candidate mediator of many tissue-specific alternative splicing events, that binds specifically to UGCAUG motifs in conserved proximal intron 16 so as to enhance splicing of E16. Major aims of the proposal include (1) characterization of the Fox-2 intron enhancer region, including affinity purification of antagonistic or synergistic co-factors that regulate E16 splicing; (2) mechanistic exploration of a uniquely distal intron conserved element, 2kb downstream, that strongly influences E16 splicing; (3) investigation of the role of Fox-2 in stage-specific splicing switches of newly identified target exons in other erythroid genes, to provide new insights into the broader erythroid splicing program; and (4) initiation of a new effort to characterize functionality of cis-regulatory elements and trans- splicing factors in vivo with animal models. In addition to its biological importance for erythroid function, the exon 16 splicing switch is one of the best models for analysis of tissue-specific splicing in any cell system. Several of these aims are novel in that relatively little is known about regulation of tissue-specific splicing by Fox-2, distal splicing regulatory elements have rarely been considered, and few studies of regulation in vivo have been reported. Successful accomplishment of these objectives will lead to a better understanding of the stage-specific switch in 4.1R pre-mRNA splicing, as well as the role of Fox-2 in the larger erythroid splicing program. These studies should also provide insights into disease mechanisms caused by aberrant splicing, ultimately leading to splicing therapeutics to correct such defects.